We have identified an enzyme activity that introduces superhelical turns into covalently closed circular DNA. This enzyme, DNA gyrase, has been extensively purified from E. coli cells. The reaction requires ATP as cofactor. The DNA product is negatively supercoiled; this is the same direction of supercoiling found in all covalently circular DNA isolated from cells. DNA gyrase is specifically inhibited by coumermycin and oxolinic acid. In both cases, enzyme from a drug-resistant mutant strain is insensitive to the drug. The two drug-resistance loci are genetically distinct, which leads us to conclude that each locus is responsible for one subunit of the enzyme. As both drugs are known to function as inhibitors of DNA replication, DNA gyrase must play an essential role in this process. BIBLIOGRAPHIC REFERENCES: Gellert, M., Mizuuchi, K., O'Dea, M.H. and Nash, H.A.: DNA gyrase: An enzyme that introduces superhelical turns into DNA. Proc. Nat. Acad. Sci. U.S.A. 73: 3872-3876, 1976. Gellert, M., O'Dea, M.H. Itoh, T. and Tomizawa, J.: Novobiocin and coumermycin inhibit DNA supercoiling catalyzed by DNA gyrase. Proc. Nat. Acad. Sci. U.S.A. 73: 4474-4478, 1976.